PROJECT SUMMARY/ABSTRACT Multiple myeloma (MM) is a hematologic malignancy of bone marrow plasma cells that is almost universally fatal. New therapies have improved survival and enabled initial disease control in almost all patients. Despite this progress, relapse with eventually fatal disease remains nearly inevitable. New treatments that specifically target the mechanisms of relapse have potential to amplify the incremental survival gains achieved with modern MM therapy. This proposal furthers a long-term goal to develop an immunotherapy that prevents MM relapse by targeting the stem-cell antigen Sox2. Prior studies and preliminary data show that post-treatment residual MM cells depend on Sox2 for survival in vivo and that anti-Sox2 immune responses may prevent development of MM from its indolent precursor condition MGUS. In a pilot clinical trial where anti-CD19 CAR T cells were used to target rare clonogenic MM cells, durable anti-Sox2 B and T cell responses were observed specifically in an extraordinary responder who is clinically MM-free five years post-treatment despite <100 days of in vivo CAR activity (Garfall et al NEJM 2015; Garfall et al JCI Insight 2018). This result is likely explained by CAR-induced immunogenic cell death, leading to durable and clinically active anti-Sox2 immunity. The hypothesis underlying this proposal is that anti-Sox2 T cells can prevent MM relapse by constraining or eliminating clonogenic Sox2- expressing MM cells that persist after cytoreductive therapy. To test this hypothesis, Sox2-specific T cells will be isolated from MGUS patients and long-term responders to CAR T cells (anti-BCMA and/or anti-CD19), using clinical data and specimens from our practice and ongoing MM clinical trials. Anti-Sox2 T cells will be character- ized for peptide and HLA specificity, and paired anti-Sox2 TCR?? heterodimers will be cloned, sequenced, and functionally validated for Sox2 reactivity in vitro. Promising TCRs will be nominated for in vivo validation based on their in vitro antigen reactivity and HLA restriction. MM patient-derived xenografts (PDX) will be generated in MISTRG6 mice, which support multilineage human bone marrow engraftment and are uniquely suited to the microenvironment dependencies of MM PDXs. Adoptively transferred T cells transduced with anti-Sox2 TCRs will be tested in PDXs for their ability to prevent secondary PDX engraftment as a model of clonogenic growth required for MM relapse. Anti-Sox2 TCRs with validated ability to prevent MM relapse in preclinical models would be candidates for clinical translation as TCR-engineered cellular therapies; likewise, validated antigenic Sox2 peptides could be translated to vaccine therapies. These candidate therapeutics would have broad potential applications in MM and other cancers where Sox2 has emerged as an important oncogene. This collaborative proposal assembles access to unique clinical samples, specialized capabilities in human T cell biology and can- cer immunology, and expertise to rapidly translate findings to a phase 1 clinical trial.